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Image Analysis

This module enables you to create automatic measurement routines very easily. The Image Analysis wizard guides you through the steps to create an automatic measurement program. It allows you to set up even complex measurement tasks easily. The steps of the wizard include image segmentation, object separation and measurement of geometrical or intensity features. After you have completed the setup, you can apply these settings to the data to be analyzed and obtain precise measurement results. You can display the results in table and list form and export them to csv-format.

For more information, see the following examples:

See also the following descriptions:

Creating a New Image Analysis Setting

When creating a new analysis setting for your images, you can select the following segmentation methods:

  • Segment Region Classes Independently: This method allows you to define several classes and subclasses. With this method, you can define the segmentation algorithm for each class independently.
  • ZOI (Zones of Influence): This method constructs a zone of influence (ZOIs) and a ring around each primary object. The primary objects are generated by segmenting the selected image channel with the selected class segmenter. The ring is defined by its width and distance from the primary object. The distance from the ZOI border from the ring can be specified. The ZOI area also incorporates the primary object and ring area.
  1. On the Analysis tab, in the Image Analysis tool, click and select New from the dropdown.
  2. In the Settings field, enter a name for your image analysis setting and click Save.
  3. A new *.czias file is created and saved in the ...\ZEN\Documents\Image Analysis Settings folder.
  4. You have created a new image analysis setting. As default the method Segment region classes independently is used.
  5. For Method, click .
  6. The Segmentation Method Selection dialog opens.
  7. From the Method drop down menu, select a method and click OK.
  8. You have created a new image analysis setting using the method of your choice.
  9. In the Image Analysis tool, click Edit Image Analysis Setting.
  1. The Image Analysis Wizard opens with the Classes step and includes already a predefined set of classes, depending on the selected method. Follow the steps in the wizard to define your image analysis. Each method comes with a predefined set of steps which allows you to make all necessary settings for image analysis.
  • Classes: Allows you to add classes and subclasses.
  • Frame: Allows you to define a measurement frame. Only the area of the frame will be analyzed.
  • Region Filter: Allows you to define simple or complex conditions to filter the detected objects according to their parameters.
  • Automatic Segmentation: Allows you to set the parameters for the automatic segmentation.
  • Interactive Segmentation: Allows you to modify the results of the automatic segmentation or draw/delete objects. Note: this step only generates relevant results in Start Interactive Analysis run.
  • Features: Allows you to select measurement features from an extensive list and to define measurement features for classes and subclasses independently.
  • Statistics: Allows you to define custom statistical features for your regions or objects.
  • Results Preview: Shows a preview of your measurement results for the current view port.

For more information, see the following examples:

Counting Number of Fluorescence Signals per Nuclei

This topic will show you how to set-up a measurement program using the Image Analysis Wizard. After this the program will be used to count the number of fluorescence spots in a multichannel image.
In this example we are using a multichannel image with 2 channels (1st channel blue (DAPI), 2nd channel green (GFP)) of fluorescence-stained cell nuclei. First, we detect the blue-stained cell nuclei in the first channel and then the green stained signals in the second channel. Then we measure the number of green fluorescence signals per nucleus.

Step 1: Classes

  1. You have created a new image analysis setting with the Segment region classes independently method and opened the Image Analysis Wizard, see Creating a New Image Analysis Setting.
  1. In the Classes step, click on Classes1 in the list and enter Nuclei in the Name input field.
  2. Select a blue color from the dropdown list in the Color section.
  3. Click on the Class 2 entry in the list and enter Individual Nucleus in the Name input field.
  4. Click on the blue channel icon in the Channel section.
  5. For Object Color, click Fixed and select the blue color from the dropdown list.
  6. You have now setup a class pair for the nuclei.
  7. Click Add Subclass.
  8. Click on Classes 3 in the list and enter Signals in the Name input field.
  9. Select a green color from the dropdown list in the Color section
  10. Click on the Class 4 entry in the list and enter Individual Signal in the Name input field.
  11. Click on the green channel icon in the Channel section.
  12. For Object Color, click Fixed and select the green color from the dropdown list.
  13. You have now setup a subclass for the signals inside the individual nucleus class (parent class).
  14. Click Next.

Step 5: Interactive Segmentation

  1. Deactivate the Interactive checkbox.
  2. Click on Next.

Step 6: Features

  1. Click on the Nuclei entry in the list.
  2. Click on the Edit button in the Regions Features section.
  3. The Feature Selection dialog is opened.
  4. Double-click in the right-hand list on the ID.
  5. The features are displayed in the Selected Features list on the left.
  6. Remove superfluous features from the list. Select the feature and click on the Delete button.
  7. Click on the Individual Nucleus entry in the list.
  8. Click on the Edit button in the Features of individual regions section.
  9. The Feature Selection dialog opens.
  10. Double-click in the right-hand list on the ID of the parent.
  11. The features are displayed in the Selected Features list on the left.
  12. Remove superfluous features (e.g. Area, Perimeter) from the list. Select the feature and click on the Delete button.
  13. Click on the Signals entry in the list. This list contains the statistical features of the individual signals.
  14. Click on the Edit button in the Regions Features section.
  15. The Feature Selection dialog opens.
  16. Double-click in the right-hand list on the ID, Count.
  17. The features are displayed in the Selected Features list on the left.
  18. For the newly added feature Count, open the Copy drop-down menu and select Copy to the parent single region.
  19. The feature is copied to the results table of the parent, in this case the individual nucleus.
  20. Remove superfluous features from the list. Select the feature and click on the Delete button.
  21. Click on the Individual Signal entry in the list.
  22. Click on the Edit button in the Features of individual regions section.
  23. The Feature Selection dialog opens.
  24. Remove superfluous features (e.g. Area, Perimeter) from the list. Select the feature and click on the Delete button.
  25. Click on the OK button.
  26. The selected features are displayed in the Regions Features section.
  27. Click on Next.

Step 7: Results Preview

  1. Click on Nuclei in the list.
  2. The number of measured nuclei is displayed in the data table to the right of the image.
  3. Click on Individual Nucleus in the list.
  4. The ID of the parent, the ID and Signals Count (the number of green signals) of the measured nuclei is displayed in the data list to the right of the image.
  5. Click on Signals in the list.
  6. The ID of the parent (corresponds to the ID of the nucleus) and the number of measured signals are displayed in the data table to the right of the image.
  7. Click on the Finish button.
  8. This saves the measurement program.

Measuring Mean Fluorescence Intensity on a Ring around the Primary Object

The following example shows how to use the Zone of Influence (ZOI) method to measure intensities within a ring that is associated to the main object, e.g. the cell nucleus. An application example are transport assays where the intensities of a certain fluorescent marker in the cytoplasm are compared to the intensities within the nucleus.

In this example we use a multichannel image of fluorescence-stained cells. The cell nuclei are stained with AF568 and the mitochondria are stained with AF488. First, we detect the nuclei in the AF568-channel as primary object. A zone of influence is generated around each detected primary object. In this area, we can define a ring and specify its thickness and distance from the main object. You can use this ring to measure intensities or to detect further sub-objects on it. For more information, see Counting the Number of Objects in a Ring around the Nucleus.

Counting the Number of Objects in a Ring around the Nucleus

This example is similar to Measuring Mean Fluorescence Intensity on a Ring around the primary Object and also uses the same data. This example shows how to count the number of objects on a ring that is associated with the main object, e.g. the cell nucleus. The images are taken from AF568 stained nuclei. The mitochondria are stained with AF488. The channel of the nuclei is used for image segmentation. The ZOI-segmentation method attributes a zone of influence (ZOI) and a ring to each detected nucleus. This area is used as a search range to detect subobjects, in this case the mitochondria.

Creating a Measurement Data Table

  1. Click on the Create Measurement Data Table button on the Analysis tab.
  2. The two data lists are now separate documents.
  3. Save each of the data lists via the File menu > Save As. Allocate a name and select .csv as the file type.
  4. The measurement data tables are saved in CSV format and can therefore be opened directly in Excel.
  5. Click on the image and save it via the File menu > Save As. Allocate a name and select .czi as the file type.
  1. The image is saved with the measurement results. If you open the image, the measurement results can be viewed in the Analysis View.

Creating an Image Analysis Setting from an Analyzed Image

It is possible to create or extract an image analysis setting from an image which has already been analyzed. This allows you to ensure that a new data set is analyzed exactly in the same way as a previously analyzed data set.

  1. You have an already analyzed image.
  1. Open your already analyzed image in ZEN.
  2. On the Analysis tab, in the Image Analysis tool, click on Options .
  3. In the drop-down list, select Create setting from analyzed image.
  4. Type in the name you want to give the analysis setting.
  5. Press Enter on your keyboard or click on .
  1. You have now created and saved an image analysis setting from an already analyzed image.

Performing an Interactive Analysis

The Image Analysis tool allows you to perform an analysis interactively. It runs the selected analysis setting with all the steps that have been marked as interactive in the setup. Steps that you have not marked as interactive in the Image Analysis Wizard are run with the values predefined in the image analysis setting. The setting does not pause to allow you to change these values interactively.

Start Interactive Analysis also allows you to directly execute an image analysis on a czi image without predefining an image analysis setting. For that you need to have an image analysis setting where all steps are marked as interactive. Then it is possible to modify every step of the Image Analysis Wizard during the interactive analysis and do a one-time image analysis on the dataset without creating a new setting. In order to retrieve an image analysis setting from an already analyzed dataset, see Creating an Image Analysis Setting from an Analyzed Image.

Note: When you analyze an image interactively, the modifications of the settings during the interactive analysis are not saved.

  1. You have defined an image analysis setting where all analysis steps you want to adjust interactively are marked as interactive.
  1. On the Analysis tab, open the Image Analysis tool.
  2. For Setting, select your image analysis setting.
  3. Click on Start Interactive Analysis.
  4. The Image Analysis Wizard opens with all the steps that are defined as interactive in the setting.
  5. Modify your settings for each step and click Next to get to the following step in the wizard.
  6. At the end, click Finish to close the wizard.
  1. You have now analyzed your image interactively and the results of this analysis are displayed.

Creating Custom Features

  1. You are in the Features step of the image analysis wizard.
  1. Select a class in the list and click Custom Feature.
  2. The Custom Feature Editor opens. All already defined features are displayed in the list, or on initial opening an empty default entry is already created.
  3. In the Custom Features list, click to add a new entry. Alternatively, if no feature has been defined yet, select the automatically displayed default entry.
  4. A new entry is added to the list.
  5. Under Define Custom Feature, define the Name for your feature and optionally specify a Unit, if applicable.
  6. In the Define Operands list, click to add a new operand. Alternatively, if no feature has been defined yet, select the automatically displayed default entry
  7. A new operand entry is created.
  8. Select the Class which is used to generate the operand.
  9. In the Features dropdown list, select the measurement feature that you want to use to define the operand.
  10. The selected class and measurement feature are displayed as Expression.
  11. Repeat the previous steps to define all operands you need to calculate your custom feature.
  12. All defined operands are displayed in the Define Operands list.
  13. Under Define Custom Expression, enter your operands and use the mathematical operators to define the calculation for your custom feature, e.g. 100*(a/b+Math.Pow(c,2)).
  14. Click Verify Expression.
  15. The syntax of your expression is checked and verified. In case the expression is not valid, an error message is displayed.
  16. Repeat this whole workflow to create all custom features required for your image analysis.
  17. All created features are displayed in the Custom Features list of the respective class.
  18. Click OK.
  19. The editor closes and saves the defined custom features. They are displayed in the list of the Features step of the wizard.
  20. After analyzing an image with the setting, the custom features are displayed in the result table of the respective class and are also available for the charts in the Analysis view, just as for any other features.

Creating Custom Statistical Features

  1. You are in the Statistics step of the image analysis wizard.
  1. Select a class in the list and click Define Custom Feature.
  2. The Custom Statistic Feature Editor opens. All already defined features are displayed in the list, or on initial opening an empty default entry is already created.
  3. In the Custom Features list, click to add a new entry. Alternatively, if no feature has been defined yet, select the automatically displayed default entry.
  4. A new entry is added to the list.
  5. Under Define Custom Feature, define the Name for your feature and optionally specify a Unit, if applicable.
  6. In the Define Operands list, click to add a new operand. Alternatively, if no feature has been defined yet, select the automatically displayed default entry
  7. A new operand entry is created.
  8. Select the Class which is used to generate the operand.
  9. In the Features dropdown list, select the measurement feature that you want to use to define the operand.
  10. Select the Statistical Operation the operand is used for.
  11. The selected class and measurement feature are displayed as Expression.
  12. Repeat the previous steps to define all operands you need to calculate your custom statistical feature.
  13. All defined operands are displayed in the Define Operands list.
  14. Under Define Custom Expression, enter your operands and click on the mathematical operators to define the calculation for your custom statistical feature, e.g.
    100*(a/b+Math.Pow(c,2)).
  15. Click Verify Expression.
  16. The syntax of your expression is checked and verified. In case the expression is not valid, an error message is displayed.
  17. Repeat this whole workflow to create all custom statistical features required for the image analysis.
  18. All created features are displayed in the Custom Features list of the respective class.
  19. Click OK.
  20. The editor closes and saves the defined custom features. They are displayed in the list of the Statistics step of the analysis wizard.

Creating an Image Analysis Setting From an AI Model

Once you have downloaded an AI model, you can use it for image analysis by creating an image analysis setting.

  1. You have downloaded or imported an AI model trained on one or multiple channels, see Downloading AI Models or Importing AI Models.
  1. On the Applications tab, open the AI Model Store tool and click Open AI Model Store. Alternatively, click for a model in the Available Models list and select Create Analysis Setting.
  2. The AI Model Store dialog opens to display all available AI models.
  3. Select the model in the list.
  4. The Properties section on the right displays more detailed information about your selected model.
  5. In the Properties section, click Create Analysis Setting.
  6. A file browser opens.
  7. Enter a name for the setting and click Save.
  8. The setting is saved as *.czias file in the respective folder.
  1. You can now use the setting for image analysis in ZEN and select it in the Image Analysis tool. Note that some parameters are pre-defined in this setting based on the model and cannot be changed, e.g. the number of classes and the segmentation method. If you need to create a more complex hierarchy level of classes (e.g. define sub-classes or Zone-of influence), set up an ordinary image analysis setting. In the Automatic Segmentation step of the setup, you can then select models trained on single channel images for the segmentation of the individual classes.

Analysis View

Only visible if an image analysis has been performed.

The Analysis view displays the following:

  • the image from the analysis
  • the table containing the analysis results
  • a scatter chart or histogram

  1. To highlight the row of the table containing the measured values of an object, click on a segmented object in the image or in the chart. To highlight multiple rows, press Ctrl and click on multiple object/ data points.
  2. To highlight the corresponding segmented object in the image, click on a row in the table or on the data point in the chart. To highlight multiple objects, press Ctrl and click on multiple rows/ data points.
  3. To highlight the measured value of an object in the scatter chart or in the histogram, click on one or more rows in the table. The corresponding data point in the chart turns red. To change the chart type, on the Custom Chart tab, click on the corresponding Chart Type button. To highlight multiple objects, press Ctrl and click on multiple rows/ objects.

You can also move your stage in the Analysis view if you click on the Stage button in the Dimensions tab and then on a position in the image. The stage is displayed as a red crosshair. This allows you to move your stage to particular points of interest which your analysis detected but would have been very hard to identify in the original image in the 2D view.

CAUTION

caution

Risk of Crushing Fingers

The drive of a microscope stage with a motorized horizontal stage axis (stage drive) is strong enough to crush fingers or objects between the stage and nearby objects (e.g. a wall).

  1. Remove your fingers or any objects from the danger area before moving the stage drive.
  2. Release the joystick immediately to stop the movement.

Examples for Custom Features

The following examples illustrate the functionality of creating custom features in the image analysis wizard, see also Creating Custom Features.

Examples for Custom Statistical Features

The following examples illustrate the functionality of creating custom statistical features in the image analysis wizard, see also Creating Custom Statistical Features.

Image Analysis Tool

Note that this tool is available only when you have licensed functionality for Image Analysis or 3D Image Analysis.

Parameter

Description

Setting

Selects the image analysis setting.


Options

Opens the options menu.

–

New

Creates a new analysis setting. Enter a name for the setting.

–

New from Template

Creates a new setting based on an existing setting. The template setting will not be modified.

–

Create new from analyzed image

Reads out czias settings from a previously analyzed image and creates a new setting from that.

–

Rename

Enables you to enter a new name for the setting.

–

Save

Saves a modified setting under the current name. An asterisk indicates the modified state.

–

Save As

Saves the current setting under a new name.

–

Import

Opens a file browser to import an existing setting.

–

Delete

Deletes the current setting.

Method

Displays the defined segmentation method for the currently selected Setting.

–


Edit

Opens the Segmentation Method dialog.

Edit Image Analysis Setting

Opens the Image Analysis Wizard to define a new analysis program or to change an existing program, see Image Analysis Wizard.

Start Interactive Analysis

Runs the selected analysis setting with all the interactive steps.
Note that steps which you have not marked as interactive in the Image Analysis Wizard are run with the values set in the analysis setting. The program does not stop to allow you to adapt them interactively.

Start Analysis

Runs the selected analysis setting without interruption.

Image Analysis Wizard

This wizard guides you through the setup of an image analysis. It is only available if you have licensed functionality for Image Analysis or 3D Image Analysis. Note that some of the parameters differ depending on whether you set up a 2D or a 3D analysis.

The following basic controls enable you to move through the steps:

Parameter

Description

Next

Moves on to the next step of the wizard.

Back

Moves back to the previous step of the wizard.

Cancel

Cancels the wizard. No changes are applied to your settings.

Finish

Saves the setup and the changes based on your progress and closes the wizard.

Measurement Features

Coordinate System

Some feature descriptions contain images with a coordinate system for illustrative purposes. Note that the actual coordinate system in the software is different and has its point of origin (0/0) in the top left corner of the image.

The software can automatically detect and measure various properties of objects.

Some general terms relevant for several feature descriptions are the following:

  • Filled: A measurement feature with Filled in its name takes the entire region for the respective calculation, i.e. any holes the region might contain are included in the calculation.
  • Unscaled: For features that are titled as Unscaled, the scaling of the image is not taken into account for the measurement. The values returned by these features have the unit pixel.

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